Rubisco activase – Rubisco's catalytic chaperone

The current status of research on the structure, regulation, mechanism and importance of Rubisco activase is reviewed. The activase is now recognized to be a member of the AAA⁺ family, whose members participate in macromolecular complexes that perform diverse chaperone-like functions. The conserved nucleotide-binding domain of AAA⁺ family members appears to have a common fold that when applied to the activase is generally consistent with previous site-directed mutagenesis studies of the activase. Regulation of the activase in species containing both isoforms can occur via redox changes in the carboxy-terminus of the larger isoform, mediated by thioredoxin-f, which alters the response of activase to the ratio of ADP to ATP in the stroma. Studies of Rubisco activation in transgenic Arabidopsis plants demonstrated that light modulation is dependent on redox regulation of the larger isoform, providing a model for the regulation in other species. Further insights into the mechanism of the activase have emerged from an analysis of the crystal structures of Rubisco conformational variants and the identification of Rubisco residues that confer specificity in its interaction with the activase. The physiological importance of the activase is reinforced by recent studies indicating that it plays a vital role in the response of photosynthesis to temperature. Rubisco activase is one of a new type of chaperone, which in this case functions to promote and maintain the catalytic activity of Rubisco.This revised version was published online in October 2005 with corrections to the Cover Date.     

Increased sensitivity of oxidized large isoform of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activase to ADP inhibition is due to an interaction between its carboxyl extension and nucleotide-binding pocket

In Arabidopsis, oxidation of the large (46-kDa) isoform activase to form a disulfide bond in the C-terminal extension (C-extension) significantly increases its ADP sensitivity for both ATP hydrolysis and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation, thereby decreasing both activities at physiological ratios of ADP/ATP. In this study, we demonstrate that the C-extension of the oxidized large activase isoform can be cross-linked with regions containing residues that contribute to the nucleotide-binding pocket, with a higher efficiency in the presence of ADP or the absence of nucleotides than with ATP. Coupled with measurements demonstrating a redox-dependent protease sensitivity of the C-extension and a lower ATP or adenosine 5'-O-(thiotriphosphate) (ATPgammaS) affinity of the oxidized large isoform than either the reduced form or the smaller isoform, the results suggest that the C-extension plays an inhibitory role in ATP hydrolysis, regulated by redox changes. In contrast, the ADP affinities of the small isoform and the reduced or oxidized large isoform were similar, which indicates that the C-extension selectively interferes with the proper binding of ATP, possibly by interfering with the coordination of the gamma-phosphate. Furthermore, replacement of conserved, negatively charged residues (Asp390, Glu394, and Asp401) in the C-extension with alanine significantly reduced the sensitivities of the mutants to ADP inhibition, which suggests the involvement of electrostatic interactions between them and positively charged residues in or near the nucleotide-binding pocket. These studies provide new insights into the mechanism of redox regulation of activase by the C-extension in the large isoform.     

Two residues of rubisco activase involved in recognition of the Rubisco substrate

Rubisco activase is an AAA(+) protein, a superfamily with members that use a "Sensor 2" domain for substrate recognition. To determine whether the analogous domain of activase is involved in recognition of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), two chimeric activases were constructed, interchanging a Sensor 2-containing region between activases from spinach and tobacco. Spinach chimeric activase was a poor activator of both spinach and tobacco Rubisco. In contrast, tobacco chimeric activase activated spinach Rubisco far better than tobacco Rubisco, similar to spinach activase. A point mutation, K311D, in the Sensor 2 domain of the tobacco chimeric activase abolished its ability to better activate spinach Rubisco. The opposite mutation, D311K, in wild type tobacco activase produced an enzyme that activated both spinach and tobacco Rubisco, whereas a second mutation, D311K/L314V, shifted the activation preference toward spinach Rubisco. The involvement of these two residues in substrate selectivity was confirmed by introducing the analogous single and double mutations in cotton activase. The ability of the two tobacco activase mutants to activate wild type and mutant Chlamydomonas Rubiscos was also examined. Tobacco D311K activase readily activated wild type and P89R but not D94K Rubisco, whereas the tobacco L314V activase only activated D94K Rubisco. The tobacco activase double mutant D311K/L314V activated wild type Chlamydomonas Rubisco better than either the P89R or D94K Rubisco mutants, mimicking activation by spinach activase. The results identified a substrate recognition region in activase in which two residues may directly interact with two residues in Rubisco.     

A non-radioactive method for measuring Rubisco activase activity in the presence of variable ATP: ADP ratios,including modifications for measuring the activity and activation state of Rubisco

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes carboxylation of ribulose-1,5-bisphosphate, the first in a series of reactions leading to the incorporation of atmospheric CO₂ into biomass. Rubisco requires Rubisco activase (RCA), an AAA+ ATPase that reactivates Rubisco by remodelling the conformation of inhibitor-bound sites. RCA is regulated by the ratio of ADP:ATP, with the precise response potentiated by redox regulation of the alpha-isoform. Measuring the effects of ADP on the activation of Rubisco by RCA using the well-established photometric assay is problematic because of the adenine nucleotide requirement of 3-phosphoglycerate (3-PGA) kinase. Described here is a novel assay for measuring RCA activity in the presence of variable ratios of ADP:ATP. The assay couples the formation of 3-PGA from ribulose 1,5-bisphosphate and CO₂ to NADH oxidation through cofactor-dependent phosphoglycerate mutase, enolase, PEP carboxylase and malate dehydrogenase. The assay was used to determine the effects of Rubisco and RCA concentration and ADP:ATP ratio on RCA activity, and to measure the activation of a modified Rubisco by RCA. Variations of the basic assay were used to measure the activation state of Rubisco in leaf extracts and the activity of purified Rubisco. The assay can be automated for high-throughput processing by conducting the reactions in two stages.     

Rubisco活化酶的研究进展

Rubisco活化酶是近年中发现的一种可以调节Rubisco活性的酶,它能使Rubisco在植株体内条件下达到最大活化程度。Rubisco活化酶不仅具有活化Rubisco的活性,而且具有ATP水解酶活性。在ATP水解过程中,Rubisco活化酶促使各种磷酸糖抑制物从Rubisco上解离下来,恢复Rubisco活性。Rubisco活化酶的发现与研究使许多Rubisco体内活化中的疑难问题得到了阐明。本文还介绍了Rubisco活化酶的分子特性、酶作用机制以及环境因素对它活性影响等方面的最新研究进展。     

Rubisco活化酶的研究进展

Rubisco活化酶是近年中发现的一种可以调节Rubisco活性的酶 ,它能使Rubisco在植株体内条件下达到最大活化程度。Rubisco活化酶不仅具有活化Rubisco的活性 ,而且具有ATP水解酶活性。在ATP水解过程中 ,Rubisco活化酶促使各种磷酸糖抑制物从Rubisco上解离下来 ,恢复Rubisco活性。Rubisco活化酶的发现与研究使许多Rubisco体内活化中的疑难问题得到了阐明。本文还介绍了Rubisco活化酶的分子特性、酶作用机制以及环境因素对它活性影响等方面的最新研究进展。     

Two conserved tryptophan residues are responsible for intrinsic fluorescence enhancement in Rubisco activase upon ATP binding

Two species-invariant tryptophan residues at positions 109 and 250 of tobacco Rubisco activase were identified by site-directed mutagenesis as being responsible for the increase in intrinsic fluorescence upon addition of ATP, which has been previously attributed to increased self-association. Substitution of W109, which is immediately prior to a ‘P-loop’ sequence in the ATP catalytic motif, with aromatic residues (Tyr or Phe), Cys or Lys eliminated both ATP hydrolysis and the intrinsic fluorescence enhancement. Although the W109 mutants bound ATP, ATP did not provide a partial protection against proteolysis by trypsin that was observed with the recombinant wild-type enzyme. In contrast, substitution of W250 with Tyr or Phe abolished about half (44%) of the increase in intrinsic fluorescence with ATP, but had little effect on ATP hydrolysis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation or proteolytic protection with ATP. The substitution of the other tryptophan residues, W16 and W305, with phenylalanine did not significantly alter the change in intrinsic fluorescence upon addition of ATP. Therefore, W109 and W250 are the residues reporting the conformational change that increases the intrinsic fluorescence.     

Evolution of Rubisco activase gene in plants

Key message

Rubisco activase of plants evolved in a stepwise manner without losing its function to adapt to the major evolutionary events including endosymbiosis and land colonization.

Abstract

Rubisco activase is an essential enzyme for photosynthesis, which removes inhibitory sugar phosphates from the active sites of Rubisco, a process necessary for Rubisco activation and carbon fixation. The gene probably evolved in cyanobacteria as different species differ for its presence. However, the gene is present in all other plant species. At least a single gene copy was maintained throughout plant evolution; but various genome and gene duplication events, which occurred during plant evolution, increased its copy number in some species. The exons and exon–intron junctions of present day higher plant’s Rca, which is conserved in most species seem to have evolved in charophytes. A unique tandem duplication of Rca gene occurred in a common grass ancestor, and the two genes evolved differently for gene structure, sequence, and expression pattern. At the protein level, starting with a primitive form in cyanobacteria, RCA of chlorophytes evolved by integrating chloroplast transit peptide (cTP), and N-terminal domains to the ATPase, Rubisco recognition and C-terminal domains. The redox regulated C-terminal extension (CTE) and the associated alternate splicing mechanism, which splices the RCA-α and RCA-β isoforms were probably gained from another gene in charophytes, conserved in most species except the members of Solanaceae family.

     

Antisense RNA inhibition of Rubisco activase expression

Ribulose bisphosphate carboxylase (Rubisco) activase catalyzes the activation of Rubisco in vivo. Activase antisense DNA mutants of tobacco have been generated to explore the control that activase exerts on the photosynthetic process. These mutants have up to 90% reductions in activase protein levels as a consequence of an inhibition of activase mRNA accumulation. It is shown that photosynthesis, measured as the rate of CO₂ exchange (CER), is modestly decreased in plants exposed to high irradiances. The decreases in CER in the transgenic plants are accompanied by corresponding decreases in Rubisco activation, indicating that activase has a direct effect on photosynthetic rates in the antisense plants by influencing the activation state of Rubisco. It is concluded that in high light conditions, control of photosynthesis is largely shared between Rubisco and activase. Plant growth is also impaired in mutant plants that have severe reductions in activase. The inhibition of activase in the antisense plants does not have an impact on the accumulation of Rubisco large subunit or small subunit mRNAs or proteins. This indicates that the concerted expression of the genes for activase (Rca) and Rubisco (rbcL and rbcS) in response to light, developmental factors and circadian controls is not due to feedback regulation of rbcL or rbcS by the amount of activase protein.     

Potential for interactions between the carboxy- and amino-termini of Rubisco activase subunits

The subunit interactions of Rubisco activase were investigated using mutants containing an introduced Cys near the N- and/or C-terminus. Chemical cross-linking of the C-terminal and double insertion mutant produced subunit dimers and dimers plus high ordered oligomers, respectively. Fluorescence measurements with N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine showed that the environment around the introduced Cys near the C-terminus becomes more hydrophilic upon nucleotide binding. The Cys insertion mutants catalyzed Rubisco activation and ATP hydrolysis even when the subunits of the C-terminal or double insertion mutants were completely cross-linked. The results indicate that the termini of adjacent activase subunits are in close proximity and can be modified and even joined without affecting enzyme function.     

Subunit interactions of Rubisco activase: polyethylene glycol promotes self-association, stimulates ATPase and activation activities, and enhances interactions with Rubisco.

The effect of polyethylene glycol (PEG) on the enzymatic and physical properties of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase was examined. In the presence of PEG, Rubisco activase exhibited higher ATPase and Rubisco activating activities, concomitant with increased apparent affinity for ATP and Rubisco. Specific ATPase activity, which was dependent on Rubisco activase concentration, was also higher in the presence of Ficoll, polyvinylpyrrolidone, and bovine serum albumin. The ability of Rubisco activase to facilitate dissociation of the tight-binding inhibitor 2-carboxyarabinitol 1-phosphate from carbamylated Rubisco was also enhanced in the presence of PEG. Mixing experiments with Rubisco activase from two different sources showed that tobacco Rubisco activase, which exhibited little activation of spinach Rubisco by itself, was inhibitory when included with spinach Rubisco activase. Polyethylene glycol improved the ability of tobacco and a mixture of tobacco plus spinach Rubisco activase to activate spinach Rubisco. Estimates based on rate zonal sedimentation and gel-filtration chromatography indicated that the apparent molecular mass of Rubisco activase was two- to fourfold higher in the presence of PEG. The increase in apparent molecular mass was consistent with the propensity of solvent-excluding reagents like PEG to promote self-association of proteins. Likewise, the change in enzymatic properties of Rubisco activase in the presence of PEG and the dependence of specific activity on protein concentration resembled changes that often accompany self-association. For Rubisco activase, high concentrations of protein in the chloroplast stroma would provide an environment conducive to self-association and cause expression of properties that would enhance its ability to function efficiently in vivo.     

Atomic resolution x-ray structure of the substrate recognition domain of higher plant ribulose-bisphosphate carboxylase/oxygenase (Rubisco) activase

The rapid release of tight-binding inhibitors from dead-end ribulose-bisphosphate carboxylase/oxygenase (Rubisco) complexes requires the activity of Rubisco activase, an AAA+ ATPase that utilizes chemo-mechanical energy to catalyze the reactivation of Rubisco. Activase is thought to play a central role in coordinating the rate of CO₂ fixation with the light reactions of photosynthesis. Here, we present a 1.9 Å crystal structure of the C-domain core of creosote activase. The fold consists of a canonical four-helix bundle, from which a paddle-like extension protrudes that entails a nine-turn helix lined by an irregularly structured peptide strand. The residues Lys-313 and Val-316 involved in the species-specific recognition of Rubisco are located near the tip of the paddle. An ionic bond between Lys-313 and Glu-309 appears to stabilize the glycine-rich end of the helix. Structural superpositions onto the distant homolog FtsH imply that the paddles extend away from the hexameric toroid in a fan-like fashion, such that the hydrophobic sides of each blade bearing Trp-302 are facing inward and the polar sides bearing Lys-313 and Val-316 are facing outward. Therefore, we speculate that upon binding, the activase paddles embrace the Rubisco cylinder by placing their hydrophobic patches near the partner protein. This model suggests that conformational adjustments at the remote end of the paddle may relate to selectivity in recognition, rather than specific ionic contacts involving Lys-313. Additionally, the superpositions predict that the catalytically critical Arg-293 does not interact with the bound nucleotide. Hypothetical ring-ring stacking and peptide threading models for Rubisco reactivation are briefly discussed.     

Small Oligomers of Ribulose-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Are Required for Biological Activity

Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) activase uses the energy from ATP hydrolysis to remove tight binding inhibitors from Rubisco, thus playing a key role in regulating photosynthesis in plants. Although several structures have recently added much needed structural information for different Rubisco activase enzymes, the arrangement of these subunits in solution remains unclear. In this study, we use a variety of techniques to show that Rubisco activase forms a wide range of structures in solution, ranging from monomers to much higher order species, and that the distribution of these species is highly dependent on protein concentration. The data support a model in which Rubisco activase forms an open spiraling structure rather than a closed hexameric structure. At protein concentrations of 1 μm, corresponding to the maximal activity of the enzyme, Rubisco activase has an oligomeric state of 2–4 subunits. We propose a model in which Rubisco activase requires at least 1 neighboring subunit for hydrolysis of ATP.     

The absence of Rubisco activase activity in total wheat leaf extracts is recovered in the purified protein

When desalted extracts of soluble protein from dark-adaptedwheat leaves were assayed for ribulose-1, 5-bisphosphate carboxylase/oxygenase(Rubisco) activase activity in the presence of 1 mM ATP andan ATP-regenerating system, very little ATP-dependent activationof RuBP-inactivated Rubisco was found. In extracts from light-adaptedleaves a very similar pattern of Rubisco activation was observedexcept that the overall level of Rubisco activity was much lowerthan in the extracts from dark-adapted leaves. These featureswere apparent both at low (120µg per ml) and high (640µg per ml) protein concentrations. We were unable to demonstrateRubisco activase activity in crude leaf extracts. Consequently,in order to establish that Rubisco activase was present in wheatleaf extracts the wheat leaf protein was purified to homogeneity.The identity of the protein was confirmed with antibodies tothe spinach enzyme, ATPase activity and activase-mediated releaseof the inhibitor, carboxyara-binitol-1-phosphate (CA1P) fromthe tertiary Rubisco complex. The pure wheat Rubisco activaserelieved the CA1P-induced inhibition of Rubisco activity. Rubiscoactivase had no significant effect on the affinity of wheatRubisco for the substrate, ribulose-1, 5-bisphosphate (RuBP). Key words: Rubisco activase, Rubisco, regulation     

Association of Rubisco activase with chaperonin-60beta: a possible mechanism for protecting photosynthesis during heat stress

Previous studies have shown that inhibition of photosynthesis by moderate heat stress is a consequence of Rubisco deactivation, caused in part by the thermal instability of Rubisco activase. This involvement of Rubisco activase was confirmed in heat stress and recovery experiments using transgenic Arabidopsis plants. Compared with wild-type plants, photosynthesis, the effective quantum yield of photosystem II, and Rubisco activation were less thermotolerant and recovered more slowly in transgenic Arabidopsis plants with reduced levels of Rubisco activase. Immunoblots showed that 65% of the Rubisco activase was recovered in the insoluble fraction after heat stress in leaf extracts of transgenic but not wild-type plants, evidence that deactivation of Rubisco was a consequence of thermal denaturation of Rubisco activase. The transgenic Arabidopsis plants used in this study contained a modified form of Rubisco activase that facilitated affinity purification of Rubisco activase and proteins that potentially interact with Rubisco activase during heat stress. Sequence analysis and immunoblotting identified the beta-subunit of chaperonin-60 (cpn60beta), the chloroplast GroEL homologue, as a protein that was bound to Rubisco activase from leaf extracts prepared from heat-stressed, but not control plants. Analysis of the proteins by non-denaturing gel electrophoresis showed that cpn60beta was associated with Rubisco activase in a high molecular mass complex. Immunoblot analysis established that the apparent association of cpn60beta with Rubisco activase was dynamic, increasing with the duration and intensity of the heat stress and decreasing following recovery. Taken together, these data suggest that cpn60beta plays a role in acclimating photosynthesis to heat stress, possibly by protecting Rubisco activase from thermal denaturation.     

Heat sensitivity of Rubisco,Rubisco activase and Rubisco binding protein in higher plants

During the past few years the investigations concerning Rubisco and the changes of its activity and properties at elevated temperature were reconsidered with special reference to the important role of Rubisco activase and Rubisco binding protein. The major changes in Rubisco, Rubisco activase and Rubisco binding protein reported recently are presented in this review. New information on these proteins, including their changes under heat stress conditions, is discussed together with open questions.     

Characterization of the regulatory function of the 46-kDa isoform of Rubisco activase from Arabidopsis

Arabidopsis Rubisco activase was recently shown to be regulated by redox changes in the larger (46-kDa) isoform specifically mediated by thioredoxin-f [Zhang and Portis (1999) Proc Natl Acad Sci USA 96: 9438–9443]. Reduction greatly increases the activity of the 46-kDa isoform and the native protein at physiological ATP/ADP ratios. In this study we conducted additional experiments to characterize the regulation of Rubisco activase by thioredoxin-f. The K_m for both ATP hydrolysis and Rubisco activation by the 46-kDa isoform was lowered by 4 to 5-fold after reduction, but the maximum activity was increased by only 10%. Only 0.35 μM thioredoxin-f was required for a half-maximal activity change after a 10 min preincubation and activation with 1 μM was complete after 10 min. Equal amounts of 46-kDa and 43-kDa isoforms were required for a complete inhibition of the Rubisco activation activity after a reduction-oxidation cycle and assay at an ATP/ADP ratio of 3:1, whereas activity was only inhibited by 50% at a 2:1 ratio (43-/46-kDa) of the isoforms. This requirement is consistent with the fact that Arabidopsis normally contains about a 1:1 ratio of the two isoforms at both the mRNA and protein levels. Redox titrations indicated a midpoint potential of −344 mV for the 46-kDa isoform as compared to −342 mV for spinach fructose 1,6-bisphosphatase at pH 7.9, consistent with previous reports indicating that these proteins are co-regulated by light intensity in a similar manner. This revised version was published online in June 2006 with corrections to the Cover Date.     

Redox modulation of Rubisco conformation and activity through its cysteine residues

Treatment of purified Rubisco with agents that specifically oxidize cysteine-thiol groups causes catalytic inactivation and increased proteolytic sensitivity of the enzyme. It has been suggested that these redox properties may sustain a mechanism of regulating Rubisco activity and turnover during senescence or stress. Current research efforts are addressing the structural basis of the redox modulation of Rubisco and the identification of critical cysteines. Redox shifts result in Rubisco conformational changes as revealed by the alteration of its proteolytic fragmentation pattern upon oxidation. In particular, the augmented susceptibility of Rubisco to proteases is due to increased exposure of a small loop (between Ser61 and Thr68) when oxidized. Progressive oxidation of Rubisco cysteines using disulphide/thiol mixtures at different ratios have shown that inactivation occurs under milder oxidative conditions than proteolytic sensitization, suggesting the involvement of different critical cysteines. Site-directed mutagenesis of conserved cysteines in the Chlamydomonas reinhardtii Rubisco identified Cys449 and Cys459 among those involved in oxidative inactivation, and Cys172 and Cys192 as the specific target for arsenite. The physiological importance of Rubisco redox regulation is supported by the in vivo response of the cysteine mutants to stress conditions. Substitution of Cys172 caused a pronounced delay in stress-induced Rubisco degradation, while the replacement of the functionally redundant Cys449-Cys459 pair resulted in an enhanced catabolism with a faster high-molecular weight polymerization and translocation to membranes. These results suggest that several cysteines contribute to a sequence of conformational changes that trigger the different stages of Rubisco catabolism under increasing oxidative conditions.     

Exceptional Sensitivity of Rubisco Activase to Thermal Denaturation in Vitro and in Vivo

Heat stress inhibits photosynthesis by reducing the activation of Rubisco by Rubisco activase. To determine if loss of activase function is caused by protein denaturation, the thermal stability of activase was examined in vitro and in vivo and compared with the stabilities of two other soluble chloroplast proteins. Isolated activase exhibited a temperature optimum for ATP hydrolysis of 44 degrees C compared with > or =60 degrees C for carboxylation by Rubisco. Light scattering showed that unfolding/aggregation occurred at 45 degrees C and 37 degrees C for activase in the presence and absence of ATPgammaS, respectively, and at 65 degrees C for Rubisco. Addition of chemically denatured rhodanese to heat-treated activase trapped partially folded activase in an insoluble complex at treatment temperatures that were similar to those that caused increased light scattering and loss of activity. To examine thermal stability in vivo, heat-treated tobacco (Nicotiana rustica cv Pulmila) protoplasts and chloroplasts were lysed with detergent in the presence of rhodanese and the amount of target protein that aggregated was determined by immunoblotting. The results of these experiments showed that thermal denaturation of activase in vivo occurred at temperatures similar to those that denatured isolated activase and far below those required to denature Rubisco or phosphoribulokinase. Edman degradation analysis of aggregated proteins from tobacco and pea (Pisum sativum cv "Little Marvel") chloroplasts showed that activase was the major protein that denatured in response to heat stress. Thus, loss of activase activity during heat stress is caused by an exceptional sensitivity of the protein to thermal denaturation and is responsible, in part, for deactivation of Rubisco.